1. Field of the Invention
The present invention relates to the identification and purification of herpes proteases and to nucleic acid segments coding for such proteases. The present invention also relates to methods of selecting candidate substances that are able to inhibit the function of these proteases and the use of such inhibitors to detect and treat viral infections.
2. Description of Related Art
a. Viral Infections Pose Major Health Problems
Treatment and prevention of viral infections is a major medical goal. To understand the state of the art in developing methods of treating and preventing viral infections, it is important to understand the structure and function of infectious virus. A virus is a small genetic element that contains either single or double-stranded DNA or RNA and can alternate between two distinct states: intracellular and extracellular. A virus is a obligatory intra-cellular parasite that cannot reproduce by itself. In effect, the virus takes over the biosynthetic machinery of the host cell and uses it for viral synthesis. Some of the protein products of the viral DNA are special enzymes or inhibitory factors that stop host cell metabolism, but most viral-encoded proteins are used in the construction of new virions. Protein synthesis is directed by the virus to produce necessary components for its replication and packaging, e.g. the capsid. These components must be assembled in an order depending on the virus, and new particles must escape from the cell if they are to infect other cells.
The general steps of the intracellular viral replication (lytic cycle) are:
1. attachment of the virus to a host cell (absorption); PA1 2. penetration of the virus or its nucleic acid into the host cell; PA1 3. replication of the viral nucleic acid; PA1 4. production of viral proteins and other essential components; PA1 5. assembly of viral nucleic acid and protein components; and PA1 6. release of mature virion particles from the host cell. PA1 (1) Preparing a nucleic acid segment which encodes the herpes protease; and PA1 (2) Allowing the segment to be expressed in order to produce the protein. PA1 HSV herpes simplex virus PA1 CMV cytomegalovirus PA1 ORF open reading frame PA1 ICP infected cellular polypeptide PA1 DFP diisopropyl fluorophosphate PA1 TPCK L-1-tosylamido-2-phenylethyl chloromethyl ketone PA1 TLCK N-.alpha.-p-tosyl-L-lysine chloromethyl ketone PA1 PMSF phenylmethylsulfonyl fluoride PA1 EGTA ethyleneglycol-bis (.beta.-aminoethyl ether) N,N,N',N'tetraacetic acid .
The overall result of the lytic cycle is new virus particles and dead host cells because the virus has appropriated the vital forces of the host. In certain types of infection, such as that caused by herpes, there may be a latent period wherein the virus resides in the host cell.
Elucidation of viral genetic systems opens the door to investigations on the mechanisms of viral infection and replication which are not simply of academic interest, but are directed toward detection, prevention, and treatment of viral caused diseases. Host resistance to viral infection may occur through absence of a viral-receptor site to prevent attachment of the virus to the host cell; destruction of the viral nucleic acids after they are injected into the host cell, for example, by cleavage of viral nucleic acids by host enzymes; inhibition of essential viral protein synthesis; or destruction of viral proteins after their formation in the host cell.
Development of antiviral drugs to supplement natural resistance is a major commercial objective whose goal is to counteract the devastating effects of many viral infections on humans. Unfortunately, treatments available to date are inadequate for most types of virus. For example, interferons are cellular antiviral substances, low molecular weight proteins, that prevent viral multiplication. However, interferons tend to be host specific, not viral specific, and have no effect on host cells already infected. Also, they can be toxic at high concentrations.
The target of antiviral drugs may be enzymes uniquely specified by the virus. For example, a major target for attack on HIV infections which cause AIDS, is the enzyme reverse transcriptase. Inhibiting this enzyme effects blockage of viral replication. Unfortunately, resistance develops to these drugs, e.g. to AZT, and is a major limitation of such treatment. The anti-herpes simplex virus drugs currently on the market are directed against enzymes which synthesize viral DNA (e.g. acyclovir). Because of emergence of resistance to these drugs, there is considerable interest in new targets.
Production of viral proteins is of particular interest as a stage where the virus may be attacked. In the extracellular or infectious state, the basic structure of viruses consists of a nucleic acid core surrounded by proteins (nucleocapsid). Some viruses also have an envelope that is external to the nucleocapsid, and contains lipids and protein. The protein coat is called the capsid. Many different proteins may constitute the capsid, depending on the virus. Some viruses encode 3-10 proteins, others more than 200. Virus particles are called virions; their role is to protect the viral nucleic acid when transferred from the cell in which it replicated to a new host cell. After transfer to the host cells, the viral intracellular state begins, and replication of the virus is potentiated. A number of non-herpes viruses appear to express proteases with cleavage site specificity which are potential targets for therapeutic intervention. However, no such protease has previously been identified for the herpes virus, a particularly widespread infectious agent for which treatment and prevention methods are grossly inadequate.
b. The Herpes Family
The family of herpes virus includes animal viruses of great clinical interest because they are the causative agents of many diseases. Epstein-Barr virus has been implicated in cancer initiation; cytomegalovirus is the greatest infectious threat to AIDS patients; and Varicella Zoster Virus, is of great concern in certain parts of the world where chicken pox and shingles are serious health problems. A worldwide increase in the incidence of sexually transmitted herpes simplex (HSV) infection has occurred in the past decade, accompanied by an increase in neonatal herpes. Contact with active ulcerative lesions or asymptomatically excreting patients can result in transmission of the infective agent. Transmission is by exposure to virus at mucosal surfaces and abraded skin, which permit the entry of virus and the initiation of viral replication in cells of the epidermis and dermis. In addition to clinically apparent lesions, latent infections may persist, in particular in nerve cells. Various stimuli may cause reactivation of the HSV infection. Consequently, this is a difficult infection to eradicate. This scourge has largely gone unchecked due to the inadequacies of treatment modalities.
c. Herpes Simplex Virus (HSV)
Herpes simplex viruses subtypes 1 and 2 (HSV-1, HSV-2), are herpes viruses that are among the most common infectious agents encountered by humans (Corey and Spear, 1986; Whitley, 1990). These viruses cause a broad spectrum of diseases which range from relatively insignificant and nuisance infections such as recurrent herpes simplex labialis, to severe and life-threatening diseases such as herpes simplex encephalitis (HSE) of older children and adults, or the disseminated infections of neonates. Clinical outcome of herpes infections is dependent upon early diagnosis and prompt initiation of antiviral therapy. However, despite some successful therapy, dermal and epidermal lesions recur, and HSV infections of neonates and infections of the brain are associated with high morbidity and mortality. Earlier diagnosis than is currently possible would improve therapeutic success. In addition, improved treatments are desperately needed.
Extrinsic assistance has been provided to infected cells, in particular, in the form of chemicals. For example, chemical inhibition of herpes viral replication has been effected by a variety of nucleoside analogues such as 5-fluorodeoxyuridine (FUDR), 5-iododeoxyuridine, 5-iododeoxyuridine, thymine arabinoside, and the like.
Some protection has been provided in experimental animal models by polyspecific or monospecific anti-HSV antibodies, HSV-primed lymphocytes, and cloned T cells to specific viral antigens (Corey and Spear, 1986). However, no satisfactory treatment has been found.
Proteases have not been identified in the herpes viruses, but there is some knowledge of the biology of the herpes virus family. Herpes viruses are double stranded DNA viruses that replicate in host cell nuclei. The herpes virion is constituted from over 30 different proteins which are assembled within the host cell. About 6-8 are used in the capsid. The preferred host cells for herpes viruses are vertebrate cells.
The herpes simplex virus 1 (HSV-1) genome specifies an abundant capsid protein complex which in denaturing gels forms multiple bands due to different molecular weights of the component proteins. Some preliminary identification of these proteins has been reported. A set of herpes simplex virus 1 (HSV-1) capsid proteins was reported by Gibson and Roizman (1972, 1974). A genetically and immunologically related family of viral capsid proteins identified by their migration bands in denaturing gels has been designated infected-cell proteins 35(ICP35). (Braun et al., 1983, 1984) At least four major and a number of minor bands in one-dimensional denaturing polyacrylamide gels, and numerous spots in two-dimensional gels, have been reported.
Braun et al. (1984) using a panel of monoclonal antibodies exemplified by H745 reported that ICP35 proteins are processed post-translationally into at least 6 species (ICP35a,b,c,d,e,f) differing in electrophoretic mobility on SDS polyacrylamide gels. Although characterized by different molecular weights, this group of virus polypeptides are detected by the same monoclonal antibodies and are coded by a region in the HSV-1 genome. Empty capsids do not contain these polypeptides. A set of proteins possibly analogous to ICP35 was reported by Preston et al. (1983).
Nucleotide sequencing has been performed on the HSV-1 genome, and attempts, generally unsuccessful, have been made to correlate various capsid proteins to sequences of the genome. For example, it has been proposed that the ICP35 proteins are encoded by the open reading frame designated U.sub.L 26 (McGeoch, et al., 1988). In the present invention it is shown that this prediction was incorrect or at least incomplete. Crude mapping of the region encoding ICP35 was attempted by Braun et al. (1984) on the basis of the analysis of HSV-1.times.HSV-2 intertypic recombinants. These authors proposed that ICP35 is encoded by a region located between the genes specifying thymidine kinase (U.sub.L 23) and glycoprotein B (U.sub.L 27). This is not a very specific prediction because it covers an area now known to include four genes.
The present invention resulted from a successful search for a new virus target for therapy. The search began by choice of the HSV-1 ICP35 protein family as a substrate. A protease target for antiviral chemotherapy, in particular, as applied to herpes virus infections, was identified in this fashion. Methods of preparing and detecting the protease, methods for selecting inhibitors of the protease, as well as detection and treatment protocols based on inhibiting the protease, are also aspects of the present invention. The finding of similarity between the gene for the herpes protease in HSV-1 and that in human cytomegalovirus indicates that the present invention is broadly applicable to all herpes virus, including HSV, CMV, EBV and VZV.